Vol. 9, No. 1
65
short communication
The sperm chromatin structure assay (SCSA)
as prognostic factor in IVF/ICSI program
Piotr Miciński1,2, Krzysztof Pawlicki3, Ewa Wielgus3, Michał Bochenek4,
Iwona Tworkowska2
2
Novomedica, Infertility Clinic, Mysłowice; 3Department of Histology
and Embryology, Silesian Medical University, Katowice; 4National
Research Institute of Animal Production, Kraków-Balice, Poland
Received: 15 April 2008; accepted: 12 January 2009
SUMMARY
In this study 60 couples undergoing intracytoplasmic sperm injection
(ICSI) procedures were enrolled. All men were classified into two groups
regarding to the DNA fractionation index (DFI) threshold value: group
I <15% and group II ≥15%. In group I, median DFI was 4%, normal
pre-implantation embryo development was observed and eleven pregnancies were achieved. In group II, median DFI was 23% and normal
pre-implantation embryo development was also observed, but only two
pregnancies were achieved. Our results suggest that the patients included
in the assisted reproductive techniques (ART) should be diagnosed with
the SCSA test and the DFI may be related to the outcome of fertilization
process as well as to the number of transferred embryos and pregnancy.
Reproductive Biology 2009, 9, 1: 65-70.
Key words: spermatozoa, sperm chromatin, DNA fragmentation index, ICSI
Corresponding author: Novomedica, ul. Bończyka 34, 41-400 Mysłowice, Poland, e-mail:
[email protected]
1
Copyright © 2009 by the Society for Biology of Reproduction
66
SCSA in IVF/ICSI program
INTRODUCTION
Several studies imply that the conventional sperm parameters (sperm concentration, motility and morphology) measured during a routine semen
analysis are not helpful when intracytoplasmic sperm injection (ICSI) is
used because they do not identify subtle defects in sperm chromatin architecture. Poor semen quality has been associated with an increase in the
proportion of sperm with DNA fragmentation [3, 8]. Recently, the sperm
chromatin structure assay (SCSA) has been recognized as an independent
measure of the sperm quality that may have higher diagnostic and prognostic capabilities than standard sperm parameters for both in vivo and in
vitro fertilization [1].
The SCSA is a rapid flow cytometry-based measurement of DNA that
determines the percentage of spermatozoa with low, moderate or high DNA
fragmentation expressed as the DNA fractionation index (DFI). The reproductive parameters affected by an increased presence of DNA abnormalities in
ejaculated spermatozoa include fertilization, blastocyst development and pregnancy rates which are of great importance for assisted reproduction technologies
[9]. The aims of this study were: 1/ to assess the sperm DNA fragmentation in
candidates for IVF-ICSI, and 2/ to compare the embryo cleavage, implantation
and pregnancy rates with the DFI values measured by the SCSA.
MATERIALS AND METHODS
In this prospective study sixty couples (mean female age: 30.5±3.7, mean
male age: 34.4±4.1) undergoing the ICSI procedures were enrolled. All men
were classified into two groups regarding the DFI threshold value: <15 %
(group I, n= 39) and ≥15% (group II, n= 21). Including criteria were: aged
under 37 years, normal hormonal parameters, normal gynecological features
and lack of infections. Excluding criteria were: woman aged above 37 years,
abnormal gynecological and hormonal parameters. Permission was obtained
from couples of both groups involved in the ICSI fertilization program.
Pregnancies were confirmed by fetal heart rate (FHR) using USG.
Miciński et al.
67
Ovarian stimulation was achieved with conventional short or long protocols involving pituitary desensitization with GnRH analog (Decapeptyl,
Ferring, Germany) and with ovarian stimulation with recombinant gonadotropin (Gonal, Serono, Switzerland), starting doses between 225 and 300 IU.
When the follicles reached about 16-20 mm in diameter, the patients were
given LH agonist (Ovitrelle, Serono, Switzerland) and 36 hours later the
oocyte retrieval was carried out by vaginal USG-guided aspiration under
general anesthesia.
Semen quality parameters - volume, pH, sperm concentration, motility
and morphology - were determined according to the guidelines of WHO [11].
Intracytoplasmic sperm injection was performed as described previously
[12]. At 16-18 hours after ICSI, the oocytes were assessed for fertilization
(at pronuclear stage) and then 48 hours after oocyte retrieval the embryos
were classified. The optimal number of 2-3 embryos (at 8 blastomeres stage)
were transferred 72 hours (84%) or 96 hours (16%) after the ova retrieval.
The SCSA was performed using a flow cytometry as described for mammalian spermatozoa [5]. The fluorescence of green (515-530 nm) and red
(>630 nm) bands was measured by DAKO Galaxy flow cytometer. Current
statistical clinical tresholds had been established to <15% and ≥15% DFI
for normal and a decreased fertility potential, respectively. For statistical
analysis, the non-parametric Mann-Whitney U test was used. Correlation
coefficient was performed with Spearman's test. Data are presented as median and lower/upper quartiles. Statistical significance was set at p<0.05.
RESULTS AND DISCUSSION
In patients with DFI ≥15% (group II), the observed percentage of normal
spermatozoa and spermatozoa with progressive motility were significantly
lower than in group with DFI in 0-15% range (group I; tab. 1). The number
of fertilized oocytes was similar for both groups, but the number of unfertilized oocytes was higher in group II than in group I. Moreover, we found
a positive correlation between DFI and unfertilized oocytes (p<0.05). In
both groups, early embryo development did not differ and the mean number
SCSA in IVF/ICSI program
68
Table 1. Selected parameters of patients undergoing ICSI procedure
Parameters
Median
DFI (%)
Group I
Group II
n = 39
n = 21
Lower Upper
Lower Upper
Median
quartile quartile
quartile quartile
4.00
3.93
5.87
23.00*
18.44
24.75
7
5
1
4
2
6
69
59
73
59*
53
65
11
8
14
12
15
15.5
3
2
5
5*
3
7.5
5
3
6
5
3
6
No. of blastomeres
10
8
12
8
8
14
No. of transferred
embryos
3
2
3
2*
2
3
No. of pregnancies
11
Spermatozoa with
progressive motility
(%)
Morphologically
normal
spermatozoa (%)
No. of oocytes
No. of infertilized
oocytes
No. of fertilized
oocytes
2*
*statistical significance p<0.05
of embryo blastomeres was similar. In group I, 3.0 (interquartile range 2-3)
and in group II, 2.0 (interquartile range 2-3) embryos were transferred and
differences between the two groups were statistically significant (p<0.05).
In group I we noted eleven pregnancies, but in the group II only two pregnancies were achieved (p<0.05).
Our data showed that only two pregnancies were initiated when DFI
was ≥15%, suggesting that sperm DNA damage has a good predictive
value in cases of post-implantation embryo development failure. Sperm
DNA integrity, cleavage rates, embryo quality and successful establishment
of pregnancy following IVF cycles were reported in some studies [7, 10].
Agrawal and Said [2] proved that sperm DNA damage shows a negative
Miciński et al.
69
correlation with embryo quality following IVF and ICSI; however, it remains
unclear whether spermatozoa with damaged DNA can impair the process
of embryo development. The quality of sperm chromatin packing affects
pregnancy rates after IVF. Filatow et al. [5] suggest that some mechanism
may prevent an embryo from developing when the organization of genetic
material in male germinal cells is abnormal. Spermatozoa with damaged
chromatin can be morphologically normal and reveal the capability for
penetrating the oocyte but they are incapable of initiating normal embryo
development [4].
In clinical practice, especially with the usage of ICSI, treatment of male
fertility still relies on diagnosis based on the light-microscope or computeraided sperm analysis of sperm morphology and motility. Larson et al. [6] and
Zini at al.[13] suggested that sperm chromatin is an independent measure of
sperm quality that may have better diagnostic and prognostic capabilities
required for an optimum fertility status than standard sperm parameters.
The SCSA has become an important tool for assessing semen quality in the
human andrology laboratory as well as in the context of assisted reproductive
techniques (ARTs) used for treating infertile couples. Our results suggest
that patients included in the ART should be diagnosed with the SCSA test
and the DFI may be related to the outcome of the fertilization process as
well as to the number of transferred embryos and pregnancy.
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